tRNA tracking for direct measurements of protein synthesis kinetics in live cells

Nature Chemical Biology
Ivan L VolkovMagnus Johansson

Abstract

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

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Citations

Jul 13, 2019·Biochemical Society Transactions·Ilijana VojnovicUlrike Endesfelder
May 18, 2018·Nature Chemical Biology·Achillefs N Kapanidis, Mathew Stracy
Oct 26, 2018·Annual Review of Biochemistry·Johan Elf, Irmeli Barkefors
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Jan 8, 2020·Virus Evolution·Benjamin R JackClaus O Wilke
Oct 6, 2020·FEMS Microbiology Reviews·Sara Rombouts, Marcelo Nollmann
Feb 9, 2021·Frontiers in Microbiology·Mikel Irastortza-Olaziregi, Orna Amster-Choder
Feb 24, 2021·Proceedings of the National Academy of Sciences of the United States of America·Javier Aguirre RiveraMagnus Johansson
Mar 22, 2021·Journal of Molecular Biology·A Carolin SeefeldtMagnus Johansson
Apr 4, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Barry S Cooperman
Nov 9, 2018·Biochemistry·Ivan L Volkov, Magnus Johansson
Oct 1, 2021·ELife·Jean-Benoît Lalanne, Gene-Wei Li

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Methods Mentioned

BETA
fluorescence microscopy
aminoacylation
gel filtration
PCR
chip

Software Mentioned

MATLAB
uncertainSPT
track
HMM
MagicPlot
SMeagol
vbSPT
custom
Origin

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